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Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

R.A. Greenstein,1,2 Ramon R. Barrales,3,4,5 Nicholas A. Sanchez,1,2 Jordan E. Bisanz,1 Sigurd Braun,3,4 and Bassem Al-Sady1 1Department of Microbiology and Immunology, George Williams Hooper Foundation, University of California at San Francisco, San Francisco, California 94143, USA; 2TETRAD Graduate Program, University of California at San Francisco, San Francisco, California 94143, USA; 3Department of Physiological Chemistry, Biomedical Center (BMC), Ludwig Maximilians University of Munich, 82152 Martinsried, Germany; 4International Max Planck Research School for Molecular and Cellular Life Sciences, 82152 Martinsried, Germany

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochro- matin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1’s catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion. [Keywords: H3K4 methylation; Set1/COMPASS; facultative heterochromatin; gene orientation; heterochromatin spreading] Supplemental material is available for this article. Received May 8, 2019; revised version accepted October 30, 2019.

Heterochromatin is a conserved nuclear ultrastructure and functional environments on each side and countering (Rea et al. 2000) that enacts genome partitioning by re- the intrinsic propensity for heterochromatin to invade pressing transcription and recombination at repetitive se- and silence genes. Major mechanisms of boundary forma- quences and structural elements, as well as genetic tion fall into three broad classes: (1) recruitment of factors information not pertaining to the specified cell fate. that directly antagonize the opposite state (for example, Once seeded at specific sequences (Hall et al. 2002; Jia by removal of state-specific signals on chromatin) (Ayoub et al. 2004; Reyes-Turcu et al. 2011), heterochromatin is et al. 2003; Schlichter and Cairns 2005; Lan et al. 2007; subsequently propagated in cis over qualitatively distinct Trewick et al. 2007; Braun et al. 2011), (2) promotion of regions of the chromosome in a process termed spreading. the original state by either depositing or protecting such Positional regulation of heterochromatin is key to deter- signals (Wang et al. 2013, 2015; Sadeghi et al. 2015; Verrier mining and remembering cell fate decisions. Boundary re- et al. 2015), or (3) structural constraint via recruitment of gions often separate adjacent heterochromatin and DNA-binding proteins that tether heterochromatin re- euchromatin domains, reinforcing the distinct signals gions to the nuclear periphery (Bell and Felsenfeld 1999;

5Present address: Centro Andaluz de Biología del Desarrollo, Universidad © 2020 Greenstein et al. This article is distributed exclusively by Cold Pablo de Olavide de Sevilla-Consejo Superior de Investigaciones Científi- Spring Harbor Laboratory Press for the first six months after the full-issue cas-Junta de Andalucía, Sevilla 41013, Spain. publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Corresponding author: [email protected] After six months, it is available under a Creative Commons License (Attri- Article published online ahead of print. Article and publication date are bution-NonCommercial 4.0 International), as described at http://creative- online at http://www.genesdev.org/cgi/doi/10.1101/gad.328468.119. commons.org/licenses/by-nc/4.0/.

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Greenstein et al.

Kurukuti et al. 2006; Noma et al. 2006). Despite the varied the processes found in metazoans. Fission yeast form con- modalities used in boundary formation, containment is stitutive heterochromatin marked by H3K9me at centro- not absolute. This is evidenced by the observation that meres, telomeres, and the mating type (MAT) locus. boundaries can be overcome by modest dosage changes Boundary formation occurs at pericentromeric regions in heterochromatin factors (Noma et al. 2006; Ceol et al. and the MAT locus via at least two mechanisms: tether- 2011), which leads to the silencing of genes critical to nor- ing to the nuclear periphery through binding of TFIIIC pro- mal cellular function. teins to B-box element sequences in boundary regions In addition to constitutive heterochromatin found at (Noma et al. 2006) as well as specific enrichment of a centromeres, telomeres, and other repetitive sequences, JmjC domain-containing protein, Epe1 (Ayoub et al. repressed domains also form at additional genomic loca- 2003; Zofall and Grewal 2006; Trewick et al. 2007; Braun tions in response to developmental and environmental et al. 2011), which recruits additional downstream boun- signals (Wen et al. 2009; Zofall et al. 2012; Zhu et al. dary effectors. In addition to these constitutive sites, 2013). These facultative heterochromatin domains are facultative heterochromatin forms at developmentally often embedded in euchromatic regions and silence devel- regulated meiotic genes in regions surrounded by canoni- opmental genes in a lineage-specific manner (Wen et al. cal euchromatin, which are partially dependent on Epe1 2009). Resulting from response to changing stimuli, the for containment (Zofall et al. 2012; Wang et al. 2015). Us- final extent of facultative domains can change over ing the well-characterized MAT locus boundary as a mod- time, expanding to different degrees (Wen et al. 2009) el for euchromatic invasion, we found that active gene and even contracting (McDonald et al. 2011) in genomic units could repel spreading and that this function depends space, though how this is achieved is not well understood. on the H3K4 methylase complex Set1/COMPASS. Set1 is Facultative domain size may be tuned at the level of the the catalytic subunit of COMPASS and is responsible for heterochromatin spreading reaction (Hathaway et al. monomethylation, dimethylation, and trimethylation of 2012) and/or the activities promoting its containment or H3K4 in vivo. It is recruited by RNA polymerase and disassembly. While little is known about the former, sev- forms a characteristic pattern of H3K4 methylation states eral models, beyond those known to operate at constitu- over genes, with H3K4me3 near the transcription start tive boundaries (Guelen et al. 2008; Zofall et al. 2012), site (TSS) and H3K4me2 in the gene body (for review, could be invoked to explain the latter. see Shilatifard 2012). We show that rather than acting as How might euchromatin regulate heterochromatin a global antagonist of spreading, like Epe1 or the histone spreading at facultative sites or respond to its expansion acetyltransferase Mst2 (Wang et al. 2015), Set1 regulates beyond constitutive domains? One of the defining fea- spreading at gene-rich environments such as hetero- tures of euchromatin is the presence of active genes. It is chromatin islands. Set1 does not exert its euchromatin thought that transcription from active genes is incompat- protective function by modulating steady-state transcript ible with heterochromatin formation (Scott et al. 2006). levels. Rather, it acts via two separate mechanisms, both Multiple direct effects of transcription have been pro- dependent on its catalytic activity: (1) the disruption of posed to interfere with heterochromatin assembly. These nucleosome stability and (2) catalytic inhibition of the include nucleosome turnover (eviction) by transcribing sole fission yeast H3K9 methylase Suv39/Clr4, by the polymerase, formation of nucleosome-depleted regions Set1 product H3K4me. This study provides a mechanism at transcriptional units, or steric interference by trans- for the encoding of spatial cues within euchromatin that cription-associated complexes (Noma et al. 2006; Garcia contain heterochromatin expansion. et al. 2010; Aygün et al. 2013). Furthermore, we under- stand that unique molecular signatures characterize eu- chromatin and heterochromatin states and are critical to Results their formation. Heterochromatin is marked by methyla- Genes can function as a barrier to heterochromatin tion of histone 3 at lysine 9 or lysine 27 (H3K9me and spreading H3K27me, respectively) and hypoacetylation of various histone lysine residues. In contrast, euchromatin features To investigate heterochromatin invasion into euchroma- H3K4me, H3K36me, and histone hyperacetylation (Niel- tin, we used our previously described heterochromatin sen et al. 2001; Guelen et al. 2008). Multiple studies have spreading sensor (HSS) (Al-Sady et al. 2016; Greenstein documented the apparent mutual exclusion of H3K9me- et al. 2018) in the euchromatic region proximal to the and H3K4me-marked regions (Litt et al. 2001; Noma MAT inverted repeat right (IR-R) boundary (Ayoub et al. et al. 2001; Cam et al. 2005; Guelen et al. 2008) and the 2000). This HSS system contains two central components: requirement for removal of signals associated with the op- (1) the spreading sensor, a monomeric Kusabira-Orange 2 posite state (Lan et al. 2007; Li et al. 2008). While we are fluorescent protein driven by the validated ade6 promot- beginning to understand how this dichotomy is formed, er, hereafter referred to as “orange,” integrated 0.7 kb out- it still remains unclear whether this is a cause or conse- side IR-R, and (2) the control, a E2Crimson fluorescent quence of separating heterochromatin and euchromatin. protein driven by the same promoter, hereafter referred We aimed to investigate the role of euchromatic signals to as “red,” integrated at a constitutive euchromatic locus in regulating the extent of spreading in fission yeast, a (Fig. 1A; Supplemental Table 1; Greenstein et al. 2018). well-characterized model system for the study of hetero- The IR-R is a well-described boundary system that can chromatin formation, which shares critical features with be easily manipulated (Garcia et al. 2015). Precisely

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Set1 constrains facultative heterochromatin

A Figure 1. Genes repel heterochromatin across boundaries in a manner dependent on Set1/COM- PASS. (A) An overview of the heterochromatin spreading sensor (HSS) outside the MAT locus IR-R boundary with transcriptional reporters encoding fluorescent proteins as sensor (“orange”) and control (“red”). IR-R (depicted as purple arrow) uses at least two independent pathways dependent on Epe1 and TFIIIC, respectively, to contain spreading of H3K9 methylation via Suv39/Clr4. IR-R function can be ab- B CD rogated by deletion of epe1 and removing the B-box- binding sequences for TFIIIC. (B) Histogram of “or- ange” signal in a WT boundary background normal- ized to Δclr4.(C) Histogram of “orange” signal in boundaryC (Δepe1) background normalized to the cor- responding WT (epe1+) strain. (D) Histogram of nor- malized “orange” signal in Δboundary (Δepe1 ΔB- box) background as in C.(E) Illustration depicting ge- EFG netic screen for modulators of gene-mediated hetero- chromatin repulsion. An HSS variant at the ura4 locus was crossed to ∼400 gene deletions. The result- ing strains were analyzed by flow cytometry. (F) His- tograms plotted as in C of normalized “orange” signal in nucleation-gated cells in WT and Δset1.(G)Data table. (Left) Fraction of cells that experienced silenc- ing at “orange.” Two thresholds were applied: a cut- off for nucleation at “green” and a cutoff for H IJ silencing at “orange.” Cells that met both criteria were counted as repressed. (Right) Odds ratio, calcu- lated by Fisher’s exact test, comparing the odds of be- ing the silenced “off” state for a cell in the Set1C mutant relative to wild-type populations. (H) Histo- gram of normalized “orange” signal Δset1 in the WT boundary background as in C.(I) Histogram of normalized “orange” signal in boundaryCΔset1 back- ground as in C.(J) Histogram of normalized “orange” signal in WT ΔboundaryΔset1 background as in C. All 1D histograms are plotted as the mean ± 3SD of 300 bootstrap iterations for combined data from the indicated number of biological isolates (n). Signal is normalized to the median signal from a Δclr4 or corresponding WT (epe1+) strain control to represent the maximum fluorescence in the absence of het- erochromatin (x = 1). A threshold for silencing (dashed red line) represents the mean signal of the WT strain less 2SD with the exception of F, where the threshold for silencing in nucleation+ cells was determined as mean less 1SD of the “orange” signal from the Δclr4 strain. The faction of cells below this cutoff was calculated (%off). controlling its disruption leads to an excellent model sys- partially compromised boundary (referred to hereafter as tem for identifying determinants within euchromatin boundaryC) (Fig. 1C, solid line histograms). In a fully com- that regulate heterochromatin spreading. With the HSS promised boundary, absent both epe1 and the five B-box system, we used flow cytometry to capture information sequence elements contained within IR-R (referred to from tens of thousands of single cells. We divided “or- hereafter as Δboundary) (Noma et al. 2006), we detected ange” by “red” for each cell to normalize for cell-to-cell increased silencing (Fig. 1D, dashed line histograms). transcription and translation noise, allowing us to quanti- However, even in the Δboundary background, >80% of fy heterochromatin-specific gene silencing at the “or- cells in the population fully express “orange.” Given ange” reporter over the population. this result, and the observation that H3K9me2 spreading We first examined the normalized orange fluorescence declines sharply over endogenous IR-R-bordering genes of a strain with a WT boundary (epe1+, B-box+) and detect- (Garcia et al. 2015), we wondered whether other activities ed no silencing in the population distribution (Fig. 1B), as beyond boundaries, possibly centered on active genes, re- expected (see legend for Fig. 1; Greenstein et al. 2018). We pel spreading. define a threshold for silencing as the mean of the appro- priate WT (epe1+) strain less two standard deviations Set1/COMPASS regulates genic protection from (see dashed red line). We next compromised one or both heterochromatin spreading of the pathways required for containment of spreading at IR-R (Ayoub et al. 2003; Trewick et al. 2007; Garcia In order to identify potential factors that regulate gene- et al. 2015) and assessed the effect on “orange” silencing. mediated repulsion of heterochromatin spreading, we de- Consistent with previous results (Garcia et al. 2015), signed a genetic screen to query the effect of gene dele- little silencing is detected in Δepe1 isolates harboring a tions on silencing measured via our reporters. We

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Greenstein et al. conducted the screen in the context of the HSS embedded imal locus. While there was not a major effect of Δset1 on at the euchromatic ura4 locus (Greenstein et al. 2018), reporter strains with a WT boundary (Fig. 1H), both boun- downstream from an ectopically placed RNAi-based het- daryC-proximal (Fig. 1I) and Δboundary-proximal (Fig. 1J) erochromatin nucleator (Fig. 1E; Marina et al. 2013). reporters experienced a significant increase in silencing This construct can generate spreading up to 8 kb down- in Δset1, supporting the hypothesis that Set1/COMPASS stream over two endogenous and two reporter genes (Sup- enacts a heterochromatin-protective function. plemental Fig. S1A; Greenstein et al. 2018), representing about one-third the size of the MAT locus. Importantly, Endogenous IR-R-proximal genes regulate H3K9me2 this 8-kb region is not protected by a natural cis-encoded spreading and silencing boundary, eliminating the need to remove any boundary factors, which avoids confounding global effects on In order to probe the effect of Δset1 on euchromatic inva- growth in the screen. Since nucleation is less robust at sion at heterochromatic sites genome-wide, we performed this locus than at endogenous heterochromatin domains chromatin immunoprecipitation followed by next-genera- (Greenstein et al. 2018), we exploited the presence of a nu- tion sequencing (ChIP-seq) with antibodies against cleator-proximal third reporter cassette encoding “green” H3K4me3 and H3K9me2 in WT, Δepe1, and Δepe1Δset1 at this locus. Based on this reporter, we can apply a com- strains that contained no reporters (Fig. 2A). We did not putational gate to isolate successfully nucleated cells perform H3K4me3 ChIP-seq for Δset1 isolates due to the (greenOFF) (described in Greenstein et al. 2018) and assess absence of H3K4me, which we validated by ChIP-qPCR their spreading state at the “orange” reporter, 3 kb down- (Supplemental Fig. S2A). Signal tracks for each genotype stream from “green.” In the WT background, the nucle- are plotted as mean and 95% confidence interval of two ation gated “orange” signal in this strain resembles the to four replicates. behavior seen in the Δboundary IR-R HSS strain (Fig. 1, Given that our above results show set1-dependent het- cf. F [black line] and D), exhibiting both gene silencing erochromatin containment at our reporter gene, we asked and fully expressed states. whether the removal of set1 would affect H3K9me2 We crossed this ura4-HSS background strain to a curat- spreading beyond IR-R (Fig. 2B). Unlike WT (black line), ed ∼400-gene subset of the S. pombe deletion library en- both Δepe1 (purple line) and Δepe1Δset1 (blue line) display riched for nuclear factors (Fig. 1E) and measured reporter similar and significant enrichment for H3K9me2 immedi- fluorescence from the resultant strains via flow cytome- ately next to IR-R, as seen by their closely superimposed try. For each strain, we plotted a 2D histogram of red-nor- means and confidence intervals (for clarity, we did not malized orange versus green fluorescence (Supplemental plot MAT internal traces). As distance increases from Fig. S1B) and calculated the fraction of cells that experi- IR-R, the traces begin to separate, with H3K9me2 signal enced silencing at “orange.” Silencing in this context is from Δepe1Δset1 strains exceeding that from Δepe1 and defined as the fraction of all cells that met both the green- WT. This separation is most evident over the open reading OFF criteria for nucleation (blue line) and had orange sig- frame of rpl401 (Fig. 2B, inset) and is statistically signifi- nal below the mean less one standard deviation of the cant as indicated by the separation of the 95% confidence matched Δclr4 strain (red line). bounds and the P-value analysis below the traces. Interest- Upon analysis of this data set, we noticed five genes ingly, this gene is also highly enriched for H3K4me3. This whose absence had the same characteristic effect of in- increase in H3K9me2 spreading significantly affects the creased silencing at the spreading reporter: ash2, swd1, transcript levels of genes proximal to the separation of swd3, spf1, and set1 (Fig. 1F,G; Supplemental Fig. S1B). the H3K9me2 traces in Δepe1Δset1 versus Δepe1 strains To probe the significance of increased silencing in these (Fig. 2C), but not genes either immediately by the compro- mutants, we performed a Fisher’s exact test and found mised boundary or beyond rpl401. We wanted to test the the odds of being in the “off” state for the mutants to be role of endogenous gene promoters in effecting the Set1- three to four times higher than for wild type (Fig. 1G). In dependent decline in H3K9me2 spreading and chose two contrast, this odds ratio comparing the other mutants genes, mtd1 and rpl401, around which spreading is most with Δset1 was close to 1 (Supplemental Fig. S1B), indi- strongly impaired. To do so, we first modified the original cating a similar likelihood of silencing. These genes are ade6p:HSS to express “orange” from the rpl401 promoter five members of the Set1/COMPASS complex, which cat- at the same locus (Fig. 2D). The rpl401 gene promoter ef- alyzes H3K4me and deposits H3K4me3 at active gene fectively repels spreading in the context of a compromised promoters (Miller et al. 2001; Noma and Grewal 2002; (boundaryC) (Fig. 2D, middle) or fully abrogated (Δboun- Santos-Rosa et al. 2002; Roguev et al. 2003). Of the re- dary) (Fig. 2D, bottom) IR-R boundary. However, the re- maining complex members, Δswd2 did not grow and moval of set1 (Δset1) resulted in complete rpl401p:HSS Δsdc1 was not in the screen, while Δshg1 showed no phe- repression in a Δboundary context (Fig. 2D). In the case notype, consistent with other studies, which denote it as of mtd1p, instead of inserting it at the original reporter lo- marginally associated with the complex (Roguev et al. cus, we replaced the endogenous mtd1 open reading frame 2003). All five gene deletions were validated by indepen- with “orange” to generate an mtd1p:HSS (Fig. 2E), which dent knockout in the parental reporter background (Sup- is located 2.5 kb from the edge of IR-R. Just like ade6p:HSS plemental Fig. S1B). and rpl401p:HSS at the IR-R-proximal locus, the mtd1p: Given this result, we sought to test whether the removal HSS also displays genic barrier function that is set1-de- of Set1C might have a similar effect at the boundary-prox- pendent (Fig. 2E). Thus, for all the promoters tested,

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Set1 constrains facultative heterochromatin

A D Figure 2. Set1 regulates H3K9me spreading at the IR-R-proximal region. (A) Overview of ChIP- seq experiment. (B, top and middle tracks) Input- normalized (see the Materials and Methods) ChIP-seq signal tracks and gene annotations for B the IR-R-proximal region. (Bottom track) H3K9me2 ChIP-seq data sets were independently normalized to signal from a sample containing merged data from both WT isolates. Tracks are represented as mean (line) and 95% confidence in- terval (shaded region) per genotype (WT n =2, Δepe1 n =3, Δepe1Δset1 n =4;each n represents a single colony deriving from a parental knockout for each genotype). P-value track represents re- gions above a threshold for H3K9me2 enrichment over background (gray boxes, 300-bp bins; absence of box indicates values below threshold). P-values for differences between genotypes are indicated in colors according to the scale. (C) RT-qPCR analy- sis for genes in the IR-R-proximal region. Error bars represent 1SD of three replicate cultures E from single colonies deriving from one parent iso- late. (n.s.) P > 0.05; (∗∗) P < 0.01 (t-test). (D, top) Overview of the rpl401p:HSS. (Middle) Histogram plots as in Figure 1 of normalized “orange” signal from set1+ (purple) and Δset1 (blue) rpl401p:HSS C boundaryC isolates. (Bottom) Histogram plots of normalized “orange” signal from rpl401p:HSS Δboundary isolates. (E, top) Overview of the mtd1p:HSS. (Bottom) Histogram plots of normal- ized “orange” signal from mtd1p:HSS boundaryC isolates.

formation of a spreading barrier is highly sensitive to the ods), we detected ∼10 heterochromatin islands (8 and presence of Set1. Given these results, we conclude that 13 in both WT replicates). These islands display TSS- Set1 contributes to the containment of spreading into proximal H3K4me and low to intermediate levels of the euchromatic region outside of IR-R in the case of H3K9me2 (Fig. 3B; Zofall et al. 2012). Our analysis found boundary failure. an increase in the number of known heterochromatin is- lands and novel ectopic H3K9me2 peaks (sites where WT shows no significant H3K9me2 enrichment) in both Set1 contributes to spreading containment at facultative Δepe1 and Δepe1Δset1 mutants (Fig. 3A; Supplemental but not constitutive heterochromatin Fig. S3A). However, we found that heterochromatin We next examined other constitutive heterochromatin spreading is exacerbated significantly in Δepe1Δset1 loci, centromeres and telomeres, for set1-mediated compared with Δepe1 at several sites, which include spreading effects. Broadly, Δset1 did not significantly in- several known heterochromatin islands (Fig. 3B). Impor- crease the extent of spreading already evident in Δepe1 tantly, the ability of Set1 to antagonize H3K9me hetero- at such loci. Marginally increased spreading was detected chromatin does not strictly depend on Epe1, as we could in Δepe1Δset1 beyond the boundaries of pericentromeric observe enhanced H3K9me2 enrichment at heterochro- heterochromatin on chromosomes II and III (Fig. 3A; Sup- matin islands and ectopic sites in Δset1 alone (Fig. 3C). plemental Fig. S2B), while at the right subtelomere I and at Since transcription at islands and island-proximal genes the pericentromere of chromosome I spreading was in fact is already extremely low in wild type (cf. Fig. 2C and reduced in Δepe1Δset1 relative to Δepe1 (Fig. 3A; Supple- Supplemental Fig. S3B), partly due to locally acting mental Fig. S2B,C). RNA processing pathways (Lee et al. 2013; Egan et al. Given the major role of Set1/COMPASS at genes and 2014; Sugiyama et al. 2016), it is not surprising that we the enrichment of H3K4me in canonical euchromatin observed only a mild further effect on transcript levels (Noma et al. 2001), we wondered whether Set1 might in Δepe1Δset1 (Supplemental Fig. S3B). These results regulate spreading at facultative heterochromatin sites, describe a critical role for set1 in spreading containment islands of H3K9me embedded in gene-rich euchromatin at gene-rich euchromatin with prominent H3K4me3 (Zofall et al. 2012; Gallagher et al. 2018). In our relatively peaks, but not at gene-poor constitutive heterochromatin stringent ChIP-seq analysis (see the Materials and Meth- regions.

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A Figure 3. Set1 regulates spreading at eu- chromatic heterochromatin islands. (A)A global analysis comparing H3K9me2 accu- mulation measured by ChIP-seq in Δepe1Δ- set1 and Δepe1 genotypes. The mean value of input-normalized H3K9me2 ChIP signal per 300-bp bin was calculated for each geno- type. For bins containing H3K9me2 signal above 1.5 times the global background, the

log2 ratio of Δepe1Δset1 over Δepe1 is plotted by chromosome (black line). Bins where this ratio exceeds a cutoff of three times enriched [red lines at y = ±log2(3)] in Δepe1Δset1 (blue) or Δepe1 (purple) are plotted as individual B points. Pericentromeres, centromeres, and telomeres are demarcated by red-shaded boxes. H3K9me2 merged peak calls from the WT strains are annotated in red along the chromosome. An asterisk denotes re- gions where mean signal is increased due to disproportionate enrichment in a single isolate. (B) Signal tracks analysis at euchro- matic heterochromatin islands and ectopic domains for H3K4me3 and H3K9me2 ChIP-Seqas inFigure 2B.P-valuescalculated as in Figure 2B except with 1200-bp bins. (C) H3K9me2 ChIP-qPCR measured at hetero- chromatin islands and ectopic domains in wild type (black) and Δset1 (light blue). Error bars represent 1SD from two technical repli- cate ChIPs. Replicate values are plotted as individual points.

C

Set1 functions in spreading containment independent towski and Kim 2010; Mikheyeva et al. 2014; D’Urso of regulating steady-state transcription et al. 2016). To directly test the involvement of mechanism 1, we examined the “orange” signal expressed from What might be the mechanisms by which Set1 confers bar- rpl401p, mtd1p, and ade6p in a set1+orΔset1 backgrounds rier activity to genes? We considered three pathways that in a WT boundary context (Fig. 4B). “Orange” signal was could account for this activity: (1) regulation of steady normalized to forward scatter (fsc). This parameter tracks state transcription by Set1, where altered frequency of with the size of a cell and has been used extensively to esti- RNA polymerase II passage would disrupt spreading; (2) in- mate the cell volume, which is a central parameter to nor- terference with heterochromatin spreading by the Set1/ malizeRNAandproteinbetweensinglecells.Theuseoffsc COMPASS enzymatic product, H3K4me; or (3) a nonenzy- bypasses any confounding effect Δset1 might have on our matic effect of chromatin-bound Set1, consistent with pri- ade6p-driven “red” control. We did not detect any major or reports (Lorenz et al. 2014; Mikheyeva et al. 2014). We decrease in “orange” in Δset1 isolates (Fig. 4B). We con- summarize the possible mechanisms in Figure 4A and in firmed this result by RT-qPCR analysis, where we normal- what follows we test whether and how mechanisms 1–3 ized ade6p “orange,” mtd1, and rpl401 transcripts to an contribute to the observed Set1-dependent barrier activity. act1 control (Supplemental Fig. S4A, left). In the normali- Previous reports have described both transcription- zation, we adjusted for the Δset1 effect on this act1 control activating and -repressive roles for Set1/COMPASS (Bura- (Materials and Methods; Supplemental Fig. S4A, right).

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Set1 constrains facultative heterochromatin

Together, these results argue against Set1 regulating the direct interference with Suv39/Clr4 activity. This could mean level of RNA polymerase II-mediated transcription potentially occur via two mechanisms—either by directly at these genes. Thus, mechanism 1 is an unlikely avenue impacting catalysis of H3K9 methylation by Suv39/ for the Δset1-dependent phenotype in reporter silencing. Clr4 or by disrupting the “read–write” positive feedback characteristic of histone methyl transferases. This spread- ing feedback mechanism is mediated by the binding H3K4me directly interferes with Suv39/Clr4 catalysis of Suv39/Clr4 enzyme to its own product via the chromo- Set1 is the only H3K4 methylase in fission yeast (Noma domain (CD), which stimulates the catalysis of H3K9 and Grewal 2002) and H3K4me and H3K9me appear methylation on proximal nucleosomes via the SET mutually exclusive (Noma et al. 2001). Hence, we hypoth- domain (Zhang et al. 2008a; Margueron et al. 2009; Al- esized one implementation of mechanism 2 could be Sady et al. 2013; Müller et al. 2016). Clr4-CD recognition of H3K9me has been shown to be sensitive to acetylation (ac) of the H3K4 residue (Xhemalce and Kouzarides 2010). A We tested whether the Clr4-CD’s ability to recognize H3K9me is impacted by H3K4me3. We purified the Clr4-CD (Supplemental Fig. S4B) and performed fluores- cence polarization with modified histone tail peptides. We found that the Clr4-CD has a similar binding affinity for H3K9me3 and H3K4me3K9me3 tail peptides (Sup- plemental Fig. S4C), which is recapitulated by the full- B length Clr4 enzyme (Supplemental Fig. S4D). Thus, the presence of H3K4me3, unlike H3K4ac, does not disrupt the “read–write” feedback mechanism. Our understanding of the effects of H3K4me on H3K9me catalysis by various enzymes are based mostly on endpoint analysis and have yielded conflicting results. Two previous studies using endpoint analysis indicated no obvious effect of H3K4me2 or a K4A mutation on C Suv39/Clr4 activity (Nakayama et al. 2001; Kusevic et al. 2017), yet a number of other studies document a range of effects of H3K4me2 or H3K4me3 on H3K9 meth- yl transferases, and these results do not always agree (Wang and Zhang 2001; Nishioka et al. 2002; Chin et al. 2005; Binda et al. 2010). To definitively determine any ef- fect of H3K4me2 or H3K4me3 may have on Suv39/Clr4 catalysis, we performed multiple turnover Michaelis-

D Figure 4. The gene-protective activity of Set1 is independent of mean transcription levels and is rooted in catalytic inhibition of Suv39/Clr4 by H3K4me2/3. (A) Possible mechanisms by which Set1 repels heterochromatin spreading: (1) maintaining a level of transcription that is refractory to heterochromatin invasion due to local RNA polymerase activity and associated cycles of nu- E cleosome eviction; (2) interference of H3K4me3, the Set1 prod- uct, with heterochromatin spreading; and (3) noncatalytic effect of Set/COMPASS, including its occupancy on chromatin. (B) Box and whisker plots of “orange” signal normalized to forward scatter (fsc) for rpl401p:HSS, mtd1p:HSS, and ade6p:HSS in set1 + (black) and Δset1 (blue) backgrounds. One percent to 99% of the data are included within the whiskers. Outliers are plotted as individual points. (C) Histone methyltransferase assay with Clr4-SET and H3(1–20) peptides with modifications as indicated.

Error bars represent 1SD from three replicate experiments. kcat/ KM (specificity constant) values are derived from measurement of the kcat and KM (see Supplemental Fig. S4F). (D) Cartoon over- view depicting FACS isolation of “low” and “high” Δboundary 5′ ade6p-“orange” cells followed by ChIP and RT-qPCR. (E) ChIP- qPCR data for FACS-sorted cells: H3K9me2 (top) and H3K4me3 (bottom). Amplicons for each qPCR are depicted as dumbbells on cartoon locus. Error bars represent 1SD from three technical replicate ChIPs.

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Menten kinetic analysis using N-terminal truncation of come, downstream transcriptional units therefore appear Clr4 comprising residues 192–490 (Collazo et al. 2005; to succumb to repressive H3K9me2. Thus, these data are Dirk et al. 2007), which includes the catalytic SET domain consistent with a model where encounter of a substantial (Fig 4C; Supplemental Fig. S4E). The masses of the and/or persistent H3K4me3 peak disrupts spreading, pro- H3K4me0, H3K4me2, and H3K4me3 peptides used were tecting downstream gene units. verified by MADLI-TOF analysis (Supplemental Fig. S9). We determined kcat, KM, and specificity constant (kcat/ Catalytic activity of Set1, and not chromatin recruitment KM) values (Fig. 4C; Supplemental Fig. S4F) and, interest- ingly, found that H3K4me3 and H3K4me2 reduce Clr4’s alone, underpins heterochromatin containment kcat/KM by 3.3 times and 1.8 times, respectively, relative To test the second prediction concerning catalytic activity to an H3K4me0 (WT) peptide. This derives mostly from of Set1, we constructed an allelic series of H3K4 methy- ’ an adverse effect on Suv39/Clr4 s kcat rather than on the lation Set1 hypomorphs, based on sequence alignments KM (see Supplemental Fig. S4F). We confirmed that this ef- with the Saccharomyces cerevisiae Set1 ortholog, and fect is reflected in the full-length enzyme under kcat/KM published catalytic mutants within this gene (Schlich- conditions (Supplemental Fig. S4G), where we found ter and Cairns 2005). We introduced tagless C862A C862A G852S H3K4me3 to reduce kcat/KM by 4.6 times, in good agree- (Set1 ) and G852S (Set1 ) (Fig. 5A) into S. pombe ment with the Michaelis-Menten parameters extracted Set1 within its native gene context, marked with a nour- with the SET domain. These results were confirmed seothricin resistance (NATR) gene, and produced a corre- with an independently produced set of H3K4me0 and sponding wild-type (wt-Set1) control. We produced a H3K4me3 peptides (data not shown). In conclusion, these separate set of strains where Set1C862A and Set1G852S and results demonstrate that Suv39/Clr4 catalysis (Fig. 4C; wt-Set1 version were N-terminally 2xFlag tagged and in- Supplemental Fig. S4F,G), but not its product recognition serted at the native set1 locus to test for expression by (Supplemental Fig. S4C,D), is inhibited by the presence of Western blot. N-terminally Flag tagged Set1 has been H3K4me3 and, to a milder extent, by H3K4me2. These shown to retain function (Mikheyeva et al. 2014). We results support a role for mechanism 2 and make the fol- found mutants and wt-Set1 to accumulate to similar levels lowing two predictions: First, if H3K4me3 is directly in- by two independent extraction methods (Supplemental volved in repelling spreading, genes downstream from an H3K4me3 peak are protected from heterochromatin inva- sion if the peak correlates with effective disruption of A C silencing. Second, chromatin recruitment of Set1 is insuf- ficient for barrier activity, which requires Set1’s catalytic activity.

B Protection of downstream genes by H3K4me To test the first prediction, we used fluorescence-assisted cell sorting (FACS) to isolate both repressed (“low”) and ex- pressed (“high”) populations of 5′ ade6p:HSS Δboundary cells (Fig. 4D) and then assessed their chromatin and tran- scriptional state via ChIP and RT-qPCR, respectively (Fig. D 4E; Supplemental Fig. S4H). While both populations evi- denced H3K9me2 accumulation upstream of the reporter, H3K9me2 signal could not be detected at any point beyond “orange” in the “high” cells (gray bars). This immediate drop coincides with the ade6p H3K4me3 peak in the “high” cells, and H3K4me3 is enriched at the downstream gene promoters comparable with WT levels. Consistent with this H3K4me3 distribution, transcription levels are Figure 5. Set1 catalytic activity but not its recruitment to chro- similar to the no heterochromatin (Δclr4) state. This re- matin is required for its gene protective function. (A) Diagram of sult, in conjunction with our above findings (Figs. 1I,J, Set1 constructs including WT-Set1 and two point mutations in 4C), suggest that H3K4me3 accumulation at ade6p pro- the catalytic SET domain: C862A and G852S. Constructs are ex- tects downstream transcriptional units. On the other pressed from the set1 promoter at the native set1 locus with an N- hand, the “low” population (black bars) displays high lev- terminal 2xFlag tag. (B) Licor Western blot for H3K4me3 with els of H3K9me2 at and beyond “orange,” while GAPDH loading control of whole-cell extracts from wild-type un- tagged Set1, the Δset1 parent of the 2xFlag constructs, 2xFlag- H3K4me3 is severely reduced (Fig. 4E). H3K9me2 levels wtSet1, 2xFlag-Set1C862A, and 2xFlag-Set1G852S. (C) Histo- eventually decline towards the essential rrb1 gene, con- gram plots as in Figure 1 of normalized “orange” signal from comitant with a rise in H3K4me3 enrichment. The dis- rpl401p:HSS Δboundary isolates that were transformed with ei- crepancy between the H3K4me3 signal in the “low” and ther untagged wt-Set1, Set1C862A, or Set1G852S. (D) Anti-Flag “high” populations thus eventually decreases with dis- ChIP-qPCR data in genetic backgrounds as in B. Error bars repre- tance. In cells where ade6p-localized H3K4me3 is over- sent 1SD from two technical replicate ChIPs.

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Fig. S5A). Next, we probed for H3K4me3 accumulation by Aygün et al. 2013). We first assessed nucleosome occupan- Western blot and found that both mutants show a defect in cy in set1+ and Δset1 strains by H3 ChIP in log phase H3K4me3 accumulation, with the C862A mutant show- cultures (Fig. 6D–F; Supplemental Fig. S6E), but also in ing almost no H3K4me3 by Western blot and G852S accu- G2-stalled cells to exclude cell cycle passage effects (Sup- mulating significantly reduced amounts (Fig. 5B). We next plemental Fig. S6F). Intriguingly, nucleosome occupancy moved to directly test above prediction, and found that is highly elevated at the 3′ of ade6 but remains low both Set1C862A and Set1G852S mutants are significantly im- throughout rpl401 (Fig. 6D; Supplemental Fig. S6E). Low paired in their ability to protect rpl401p:HSS from invasion nucleosome occupancy is strongly antagonistic to spread- by heterochromatin compared with wt-Set1 (Fig. 5C). ing (Garcia et al. 2010; Aygün et al. 2013). Therefore, the Set1/COMPASS, similar to Suv39/Clr4, contains a posi- data showing that ade6 retains high nucleosome occupan- tive feedback loop with the enzyme recognizing its prod- cy at its 3′, provide an additional explanation why ade6 uct (Roguev et al. 2003; Kirmizis et al. 2007), and it is and not rpl401 is vulnerable to heterochromatin invasion possible that abrogation of catalytic function leads to re- from the 3′. More broadly, we observed increases in nucle- duction of Set1 recruitment to chromatin. We used our osome occupancy at heterochromatin islands and the IR- 2xFlag Set1 constructs to test whether Set1C862A and R-proximal genes in Δset1 (Fig. 6E,F) and across active Set1G852S are still normally recruited to chromatin at the genes distributed on the three S. pombe chromosomes TSS (Fig. 5D) and indeed found no major difference in en- (Supplemental Fig. S6E), but not on heterochromatin tar- richment of Set1C862A or Set1G852S versus wt-Set1. Impor- gets (Fig. 6E, gray box). This effect therefore likely repre- tantly, this data allows us to exclude that the recruitment sents a general feature of Set1 activity. Importantly, we of Set1, part of a megadalton complex (Miller et al. 2001), found that the catalytic activity of Set1 is required for itself or associated H3K4me-independent functions, repel this regulation of nucleosome occupancy, as the catalytic heterochromatin spreading (mechanism 3) (Fig. 4A). In- Set1C862A and Set1G852S mutants partially or fully mirror stead, these data offer further support for mechanism 2, the Δset1 phenotype at ade6, rpl401, and heterochromatin showing in vivo that Set1 catalytic activity is required islands (Fig. 6D,F). The extent to which the catalytic mu- for containment of heterochromatin spreading, likely in tants recapitulate the Δset1 phenotype correlates both part via direct interference with Suv39/Clr4 catalysis. with the global H3K4me3 accumulation defect in each hypomorph (Fig. 5B), as well as the residual H3K4me3 at any given gene-internal location relative to the wt-Set1 The distribution of H3K4me3 and nucleosome (Fig. 6C,D). occupancy over genes correlate with orientation However, these data do not explain how heterochroma- dependence of genic heterochromatin boundary function tin can overcome the TSS-localized H3K4me3 peak when H3K4me3 is enriched near the TSSs of genes (Santos-Rosa invading a gene like ade6 from 3′ and then enact stable re- et al. 2002; Pokholok et al. 2005), and in fission yeast, het- pression. We hypothesized that for this to occur, 3′-invad- erochromatin silencing can proceed in a cotranscriptional ing heterochromatin would need to be able to (1) partially manner (Bühler et al. 2006, 2008). This led us to hypothe- invade the gene, (2) down-regulate transcription without size that encountering a gene first at the promoter (5′ end) fully reaching the promoter, consistent with cotranscrip- versus the terminator (3′ end) will more effectively protect tional gene silencing, and, finally, (3) reduce H3K4me3, against gene silencing, since heterochromatin will be an- the key spreading antagonizing signal, likely via a reduc- tagonized before cotranscriptional silencing mechanisms tion in transcription (Shilatifard 2012). To address these can proceed. To our surprise, while our ade6p:HSS is hypotheses, we built a variant of the 3′ade6p:HSS reporter clearly more effective in the 5′-proximal orientation (Fig. construct that would permit spreading to proceed into the 6A; Supplemental Fig. S6A), rpl401p:HSS shows much gene unit but hinder its ability to reach the promoter. To less bias (Fig. 6B; Supplemental Fig. S6B). In both 5′- and achieve this, we fused the “orange” and “green” coding se- 3′-proximal orientations, rpl401p:HSS effectively disrupts quences by an in-frame linker containing five B-box ele- spreading in a set1-dependent manner (Fig. 6B). We won- ments (Supplemental Fig. S7A), multimers of which dered whether this discrepancy can be explained by the have been shown to confer synthetic boundary activity profile of H3K4me3 over the native gene. Indeed, we found (Noma et al. 2006). Signal from “green” and “orange” in that rpl401 has significantly elevated H3K4me3 over WT, boundaryC, and Δboundary contexts, as well as their middle and, critically, 3′ of the gene, while it was strongly RNA levels (Supplemental Fig. S7A,B), were well correlat- diminished at the 3′ of ade6 (Fig. 6C). We found a similar ed in each isolate. This indicates that the entire transcrip- distribution pattern for the respective HSS cassettes tional unit is uniformly regulated, despite presence of the (Supplemental Fig. S6D). The difference in orientation synthetic B-box boundary midway through the tandem bias between ade6p:HSS and rpl401p:HSS can thus be par- gene unit. We next assessed the chromatin state at tially accounted for by the H3K4me3 profile. However, we “green” and “orange” by ChIP. H3K9me2 is significantly wondered whether additional, nonetheless H3K4me-de- reduced at “orange” compared with “green” across all iso- pendent, mechanisms beyond the direct catalytic interfer- lates from both boundaryC and Δboundary contexts (Sup- ence we document above, underlie the striking difference plemental Fig. S7C) supporting that the 5x B-box sequence in gene orientation effect. was functioning as a synthetic roadblock to spreading. We focused on regulation of nucleosome occupancy, The difficulty of separating nucleosomes by shearing known to adversely affect spreading (Garcia et al. 2010; within heterochromatin likely prevented us from

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A Figure 6. H3K4me3 disrupts local nucleo- some occupancy. (A) Locus cartoon for ade6p- driven “orange” reporters in either the 3′ or 5′ (shaded) orientation with respect to IR-R (car- toon). Histograms for normalized “orange” sig- nal in Δboundary context as in Figure 1. 3′ ade6p:HSS is plotted in full color, while 5′ ade6p:HSS is shaded (data depicted in Fig. 1, re- B drawn for comparison). epe1 and set1 geno- types are as indicated. (B) Locus cartoons and histogram plots as in A for 3′ and 5′ rpl401p: HSS. Shaded lines reproduced from Figure 2D. (C) H3K4me3 ChIP-qPCR over the gene body of ade6 and rpl401 open reading frames in wild-type untagged Set1, Δset1, 2xFlag- wtSet1, 2xFlag-Set1C862A, and 2xFlag- C Set1G852S. (D) H3 ChIP-qPCR over the gene body of ade6 and rpl401 open reading frames in genotypes as in C.(E) H3 ChIP-qPCR in WT (black) and Δset1 (blue). Constitutive het- erochromatin targets (boxed in gray) (F)H3 ChIP-qPCR at mei4, iec1, and act1 in geno- types as in C.InE H3 ChIP-qPCR Error bars represent 1SD from four replicates, each repre- D senting a single colony deriving from each ge- notype. For ChIPs in C, D, and F, error bars represent 1SD from two technical replicate ChIPs.

EF

documenting any potentially sharper drops across the syn- Previous studies have identified a role for Set1/COMPASS thetic barrier. Surprisingly, H3K4me3 ChIP revealed that and H3K4me in promoting global histone acetylation at boundaryC and Δboundary had significantly reduced various residues (Noma and Grewal 2002; Taverna et al. methylation levels compared with WT at the “orange” 2006; Ginsburg et al. 2014). To validate this finding in TSS (Supplemental Fig. S7D). These results demonstrate our system, we performed ChIP against H3K9ac, as well that invasion of a gene from the 3′ end can reduce both in- as H3 and H4 acetylation broadly, and found indeed that hibitory H3K4me levels and transcription, despite not ful- in Δset1 acetylation was similarly reduced (Fig. 7A–C) at ly reaching the gene promoter. This mechanism would all of the genes tested, whether at heterochromatin is- presumably not operate in the 5′ orientation, since lands or canonical euchromatin. The fact that we found H3K4me3 would be encountered first. a robust decrease in H3 and H4 acetylation as well as H3K9ac specifically, indicates the involvement of multi- ple histone acetyltransferase (HAT) complexes (Buratow- Histone acetylation links H3K4me3 to Set1-dependent ski and Kim 2010; Woo et al. 2017). Since HAT mutants regulation of nucleosome occupancy or knock-downs have broad effects on heterochromatin Our data show both that the catalytic activity of Set1, (Gomez et al. 2005; Tong et al. 2012; Wang et al. 2013, hence production of H3K4me, is required for containment 2015), we chose not to pursue mutational analysis of of heterochromatin spreading and that regulation of the catalytic subunits. S. pombe contains two genes that nucleosome mobilization tightly correlates with this con- are orthologs of H3K4me3-specific PHD reader modules tainment function. However, the question remains of how within HAT complexes: png1, which associates with regulation of nucleosome turnover is tied to H3K4me. Mst1 in S. pombe (Chen et al. 2010), and png2, which

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A B C Figure 7. Role of histone acetylation in hetero- chromatin containment. (A) H3 (pan) acetyl ChIP-qPCR in WT and Δset1. ChIP is normalized to H3 signal to account for differences in nucleo- some occupancy. (B) H3K9ac ChIP-qPCR plotted as in A.(C) H4 (pan) acetyl ChIP-qPCR plotted as in A. For A–C, error bars represent 1SD from four replicates, each representing a single colony deriv- ing from each genotype. (D) Cartoon depicting D Png1 and Png2 containing H3K4me3 reading PHD finger domains. Png1 is associated with NuA4.Histograms as in Figure 1of normalized “or- ange” signal from 3′ rpl401p:HSS Δboundary iso- lates from png1ΔPHD (brown), or WT boundary with png1ΔPHD (grey) and png1+ (black). (E) Model for the contribution of Set1/COMPASS to gene- mediated heterochromatin repulsion.(Top)In5′ in- E vasion, Set1-dependent TSS-proximal H3K4me3 repels heterochromatin spreading via direct Suv39/Clr4 inhibition and nucleosome destabili- zation. (Bottom) Broader distributions of Set1-de- pendent H3K4me3 in bodies of some genes and the ensuing increased nucleosome destabilization repels 3′ heterochromatin invasion. Histone ace- tyltransferase complexes (HATs) attracted to the H3K4me3 via reader proteins acetylate locally and contribute to nucleosome destabilization.

does not impact H4 acetylation or have known HAT in H3K4me-dependent heterochromatin containment, associations in S. pombe (Chen et al. 2010). To test wheth- possibly recruiting Mst1, which likely acts redundantly er recruitment of HATs to H3K4me3 specifically is in- with other HAT complexes (Buratowski and Kim 2010). volved in providing protection against heterochromatin invasion, we deleted either the H3K4me3-reading PHD ′ fingers of png1 or png2 in the context of the 3 -oriented Discussion rpl401p:HSS. While png2ΔPHD did not have an effect on rpl401p:HSS in the Δboundary context, we found Two paradigms have emerged for heterochromatin png1ΔPHD to have a subtle but highly reproducible effect, domain regulation, which when taken together present resulting in elevated silencing at the otherwise highly ef- an intriguing paradox. On one hand is the ability for het- ficient barrier forming 3′- rpl401p:HSS (Fig. 7D). This phe- erochromatin domains to expand beyond their borders notype was recovered after outcrossing to wild type and when containment mechanisms are compromised retesting nine bone fide Δepe1 png1ΔPHD resulting prog- (Noma et al. 2006; Zofall and Grewal 2006; Trewick eny (Supplemental Fig. S8A). Additionally, the phenotype et al. 2007; Zofall et al. 2012; Wang et al. 2013, 2014; was recovered upon reintroduction of Δepe1 into epe1+ Garcia et al. 2015). On the other hand is the widespread png1ΔPHD isolates, indicating it is a stable phenotype dispersion of factors, activities, and posttranslational (Fig. 7D). png1ΔPHD importantly does not affect basal ex- modifications embedded in euchromatin, which are pression of rpl401p:HSS in the presence of epe1+ (Fig. 7D, known to antagonize the establishment and maintenance dashed gray line). The fact that the phenotype is signifi- of heterochromatic domains (Lan et al. 2007; Sugiyama cantly weaker than Δset1 is expected, since it appears et al. 2007; Garcia et al. 2010; Aygün et al. 2013; that more than one HAT is involved in maintaining ele- Wang et al. 2013, 2015). Why then is heterochromatin vated acetylation in response to Set1 activity (Fig. 7A– spreading able to overcome these negative regulators C). The Δset1 png1ΔPHD double mutant has very similar and expand into euchromatin? Part of the answer may degree of silencing as Δset1, 86% versus 82% of cells, re- lie in the activities inherently associated with the spread- spectively, (Supplemental Fig. S8B), indicating that the ef- ing machinery, including HDACs (Grewal et al. 1998; fect of png1ΔPHD on silencing is likely not additive with Shankaranarayana et al. 2003; Yamada et al. 2005; Sugiya- Set1. These data provide evidence for Png1’s involvement ma et al. 2007), nucleosome remodelers (Sugiyama et al.

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2007; Taneja et al. 2017), and H3K4 – demethylase com- 2017), we found that H3K9me catalysis by Suv39/Clr4 plexes (Li et al. 2008), which apparently can overpower eu- H3K9 is directly inhibited by Set1 products, most strongly chromatin. However, how and why heterochromatin by H3K4me3. This finding represents a rare example of di- spreading is halted at specific euchromatic locations is rect regulation of the Suv39/Clr4 SET domain active site, not understood. beyond autoinhibition (Iglesias et al. 2018), but is consis- In this work we investigated the signals within local ac- tent with the effect H3K4me can have on other H3K9 tive euchromatin that define spatial limits to heterochro- methylases (Wang and Zhang 2001; Nishioka et al. matin spreading in fission yeast. The key principles that 2002; Binda et al. 2010). (2) Locally decreased nucleosome derive from this work are as follows: (1) Euchromatic bar- occupancy. We found that the distribution of H3K4me3 rier signals depend on Set1/COMPASS activity at active tracks with a Set1-dependent decrease in nucleosome oc- genes. (2) High gene transcript levels are not intrinsically cupancy. It is known that specific nucleosome stabilizing refractory to heterochromatin invasion. (3) Set1-depen- factors are required for constitutive heterochromatin as- dent repulsion of heterochromatin acts via two pathways sembly (Yamane et al. 2011; Taneja et al. 2017), and we re- downstream from H3K4 methylation: direct catalytic in- cently showed that repression of turnover is critical to hibition of Clr4/Suv39 and nucleosome mobilization. (4) stable spreading (Greenstein et al. 2018). Thus, the disrup- The ability to repel heterochromatin can be gene orienta- tion of nucleosome occupancy by Set1 will antagonize tion-specific, directed by the distribution of H3K4me over heterochromatin formation. Increased mobilization is de- the gene. pendent on Set1’s catalytic activity (Fig. 6D,F), raising the question of how increased mobilization is instructed. In principle, this could occur via direct recruitment of nucle- Mechanisms regulating facultative heterochromatin osome remodelers, or via changes in the chromatin land- domain size scape that increase nucleosome turnover. Set1 has been We found Set1/COMPASS enacts a heterochromatin con- shown to increase histone acetylation (Noma and Grewal tainment signal at gene-rich regions, including facultative 2002; Ginsburg et al. 2014), which has long been linked to heterochromatin in fission yeast that responds to environ- decreased nucleosome occupancy (Reinke and Hörz 2003; mental conditions (Figs. 2,3; Zofall et al. 2012; Sugiyama Wirén et al. 2005) and stability (Ausio and Van Holde et al. 2016; Gallagher et al. 2018). These findings are in 1986; Brower-Toland et al. 2005). We observed strong, contrast to previously identified spreading regulators Set1-dependent increases in pan-H3ac and H4ac, as well that function globally, such as Epe1, Leo1, Paf1, and as H3K9ac in most genes tested, including all those acting Mst2 (Trewick et al. 2007; Zofall et al. 2012; Kowalik as spreading boundaries in our system (Fig. 7A–C). The et al. 2015; Sadeghi et al. 2015; Verrier et al. 2015; Wang H3K4me3 HAT targeting pathways in S. pombe are not et al. 2015; Flury et al. 2017). The containment function well understood. However, the involvement of HAT tar- of Set1 is localized to specific euchromatic regions, het- geting downstream from H3K4me3 is evidenced by the erochromatin islands, and euchromatin exposed to boun- moderate loss of heterochromatin containment at dary failure at IR-R (Figs. 2,3), but is not prominent at rpl401p:HSS in the in-frame deletion of the Png1, but constitutive heterochromatin (Supplemental Fig. S2C). not Png2, PHD finger, a conserved H3K4me3 targeting Critically, containment of heterochromatin spreading module (Fig. 7D,E). This result is consistent with the ob- does not require a change in mean transcript level (Fig. servation that in S. pombe, only Png1 and not Png2 asso- 4B), but specifically its ability to methylate H3K4. This ciates with a HAT (Chen et al. 2010). We believe that this is remarkable as active transcription in fission yeast and phenotype indicates significant contribution of Png1 in other systems leads to formation of nucleosome-free re- containment given that (1). rpl401 features a very large gions (NFRs) at the TSS (Lantermann et al. 2010), and H3K4me3 peak (Fig. 2B) and the 3′-oriented HSS we NFRs are thought to be refractory to heterochromatin used is our strongest barrier construct, and (2) HATs likely spreading (Garcia et al. 2010; Lantermann et al. 2010). act additively in implementing the H3K4me3 signal, as Our data point to NFRs still being intact in Δset1, as is ev- we observed increases in H3ac and H4ac, which are ident from the coinciding dips in the H3K9me2 tracks in known to be mediated by a number of HATs including Δepe1 and Δepe1Δset1 (Fig. 2B). This is consistent with SAGA, Mst1, Mst2, and Hat1. Further, it remains possible findings that formation of NFRs alone is insufficient to that direct recruitment of chromatin remodelers by block spreading (Oki and Kamakaka 2005). Just as hetero- H3K4me works in concert with histone acetylation. Col- chromatin overcomes the TSS-proximal NFR it can over- lectively, our data point to catalytic interference and re- come the presence of Set1 on chromatin, even though it is duced nucleosome occupancy working synergistically in part of a megadalton complex. This conclusion is support- the containment of heterochromatin spread downstream ed by the normal chromatin localization, but defective of Set1. Of note, unlike in fission yeast as documented H3K4 methylation and heterochromatin repulsion, of here, in budding yeast, Set1 has a more global heterochro- the Set catalytic hypomorphs (Fig. 5D). The crucial het- matin-antagonizing role, in concert with H2A.Z (Venka- erochromatin repelling signal is, therefore, the H3K4me tasubrahmanyam et al. 2007). This suggests that Set1’s mark. This modification takes two parallel tracks to role in constraining heterochromatin in euchromatin push back against encroaching heterochromatin: (1) Cata- specifically may have coevolved with H3K9me-marked lytic interference. In contrast to prior findings obtained by heterochromatin systems, with other factors regulating endpoint analysis (Nakayama et al. 2001; Kusevic et al. constitutive domains (see above).

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Set1 constrains facultative heterochromatin

Regulation of active and repressed chromatin states The differential distributions of H3K4me and nucleosome by Set1 and COMPASS occupancy changes we observed across genes give rise to an orientation bias in the ability of a gene to repel hetero- How do the mechanisms of heterochromatin regulation chromatin (Fig. 6A). If gene orientation can influence con- we describe for Set1/COMPASS relate to its known roles tainment effectiveness, an orientation bias may emerge at in transcriptional regulation? The recruitment of Set1/ genomic sites where containment of silencing is critical. COMPASS to chromatin requires H2B monoubiquitina- Such a case has indeed been documented in mammals. tion mediated by Rad6 and Bre1 as well as interaction Lamina-associated domains (LADs) are gene-repressive with the Paf1 elongation complex (Paf1C), which engages chromatin domains associated with the nuclear periphery RNA polymerase and is additionally responsible for acti- that contain both H3K9 and H3K27 methylation (for re- vation of Rad6 and Bre1 function on chromatin. Set1/ view, see van Steensel and Belmont 2017) and regions im- COMPASS also associates with elongating RNA polymer- mediately flanking LADs are enriched for 5′ oriented ase, giving rise to a characteristic pattern of H3K4 methyl- genes and concomitant H3K4 methylation (Guelen et al. ation states (see above). Interestingly, previous studies in 2008). It is not surprising that mammalian genomes may fission yeast have described a role for Paf1C components require use of 5′ orientation more than fission yeasts, Paf1 and Leo1 in antagonizing heterochromatin spreading which lack such a bias at boundaries of constitutive het- through promoting increased histone turnover and H4K16 erochromatin domains (Supplemental Fig. S8C): Yeast acetylation (Sadeghi et al. 2015; Verrier et al. 2015). Both genes are very small, at a median length of ∼1.8 kb, with studies tested, but did not identify, a role for Set1 in their the H3K4me3 peak comprising, on average, 25% of respective systems at loci (IRC1L of the centromere and the gene, while mouse and human genes have a median IR-L of MAT), where we also do not detect an effect of length of 16 and 20 kb, respectively (Supplemental Fig. Δset1 even in the sensitized Δepe1 genetic background S8D), yet preserve a similar TSS-localized H3K4me3 (Supplemental Fig. S2). peak (Guenther et al. 2007). Thus, it is plausible that Several additional data support a model where Set1 and H3K4me3 signals distribute far enough across a gene to Paf1/Leo1 act in separate pathways to regulate hetero- make effective boundaries in either orientation for fission chromatin spreading: (1) set1 was not found to be epistatic yeast genes, but explains a 5′ bias for mammalian genes to leo1 in a genome-wide genetic interaction study for het- where the H3K4me3 peak is restricted to a narrow fraction erochromatin spreading using an IRC1L reporter (Verrier of the gene. et al. 2015). (2) Global H4K16 acetylation levels did not Our above results lead to a model (Fig. 7E) for how fac- change in response to Δset1 (Noma and Grewal 2002), ultative heterochromatin domains can be delimited in a whereas acetyl marks such as H3K9 and H3K14 were re- manner that is specific in genomic space. It remains to duced in this background (Fig. 7B; Noma and Grewal be determined why only some, but not other, euchromati- 2002). (3) In our repelling factor screen, Δleo1 did not re- cally embedded heterochromatin domains require Set1 for sult in the characteristic spreading phenotype seen for their containment, and we believe this may be encoded in Set1/COMPASS complex deletions (data not shown). Tak- the relative rates of local heterochromatin spreading and en together, these results describe separate mechanisms availability of limiting factors (Nakayama et al. 2000; for spreading regulation by Paf1/Leo1 and Set1/COM- Noma et al. 2006; Kagansky et al. 2009). Regardless, the PASS. Additionally, since the observations on heterochro- gene-centered role of Set1/COMPASS we document here matin containment are dependent on Set1’s catalytic in constraining heterochromatin spreading gives insight activity (Fig. 5), they are unlikely to be related to the into the mechanisms of locus encoded and potentially gene-repressive functions of Set1 that are independent of cell-type specific restriction of facultative gene-repressive its H3K4me catalytic activity (Lorenz et al. 2014). domains, as opposed to the global means of delimiting heterochromatin that have been described to date. The role of gene orientation in heterochromatin repulsion The nucleosome mobilizing effect of Set1 we document is Materials and methods generally strongest close to the TSS, as is evident from Strain and plasmid construction ade6, as well as the very long sib1 gene, where we ob- served lowest occupancy and Set1 dependence at the 5′ Plasmids used to generate genomic integration constructs were end (Supplemental Fig. S6E). However, in the case of assembled using in vivo recombination. S. pombe transformants rpl401, both H3K4me3 and the concomitant decrease in were selected as described (Greenstein et al. 2018). XFP reporters occupancy is much more broadly distributed. This phe- were targeted to specific genomic locations as described (Green- stein et al. 2018). Direct gene knockout constructs were generat- nomenon is especially true for H3K4me2, which is evenly ed using long primer PCR to amplify resistance cassettes with distributed throughout the rpl401p:HSS reporter (Supple- homology to the regions surrounding the open reading frame of mental Fig. S6D). While recruitment of HATs via PHD the target. Genomic integrations were confirmed by PCR. fingers is H3K4me3-specific (Li et al. 2006; Taverna et al. 2006), Suv39/Clr4 catalysis is still impacted by H3K4me2 (Fig. 4C), implying that both methylation Flow cytometry and FACS sorting states could work in concert through both catalytic and Cells were grown for flow cytometry experiments as described nucleosome mobilization pathways to repel spreading. (Greenstein et al. 2018). Flow cytometry was performed using a

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Greenstein et al.

Fortessa X20 dual machine (Becton Dickinson) and high-through- the following modifications. For Figure 4E, 16 × 106 to –18 × 106 put sampler (HTS) module. Approximately 20,000–100,000 cells cells of both “low” and “high” FACS populations, as well as con- were collected, dependent on strain growth and volume collected. trols, were collected and processed for ChIP. Prior to lysis, 50 × Fluorescence detection, compensation, and data analysis were as 106 cells of independently fixed S. cerevisiae W303 strain were described (Al-Sady et al. 2016; Greenstein et al. 2018). added to each population as carrier. ChIP experiments with For the FACS experiment, cells were grown overnight from OD bulk populations of log phase cells were performed as described = 0.05 in YES and in the morning concentrated into a smaller vol- (Greenstein et al. 2018) without the addition of W303 carrier. In ume (∼3–5×) and filtered with 35–40-µm mesh (Corning) to Supplemental Figure S6F, Hht2-HA cells were grown at 25°C achieve 5000–7000 events/sec on the cytometer and reduce po- and 225 rpm in YES + hygromycin B from OD = 0.05. After cells tential for clogs. Cells were first gated for size (forward and side reached OD = 0.2, G2 stall was induced by shifting the tempera- scatter), removal of doublet cells, and the presence of the control ture to 37°C for 3 h prior to fixation. Following lysis, sonication “red” signal and then sorted into low and high populations for was performed using a Diagenode BioRuptor Pico for 20–28 “orange.” Low “orange” population was defined by signal over- rounds of 30 sec on/30 sec rest or Diagenode BioRuptor standard lapping a control with no fluors. High “orange” population was on high for 30–40 for rounds of 30 sec on/30 sec rest. Cleared chro- defined by signal overlapping the matched background Δclr4 con- matin was split into equal volumes per IP after a small fraction trol. For each population, 16 × 106 to 18 × 106 cells were collected (5%–10%) was set aside as input/WCE. One microliter of the fol- for chromatin immunoprecipitation and 3 × 106 cells were col- lowing antibodies was added per ChIP sample: H3K9me2 lected for RT-qPCR. Cells were processed for downstream analy- (Abcam, ab1220), H3K4me3 (Active Motif 39159), H3K4me2 (Ac- sis immediately following sorting. tive Motif, 39141), H3K9ac (Active Motif, 39137), H3(pan)ac (Ac- tive Motif, 39064), H4(pan)ac (Active Motif, 39140), HA (Abcam, ab9110); 1.5 µL of anti-Flag M2 antibody (Sigma) was added per Repelling factor screen ChIP sample, and 1.4 μg of H3 antibody (Active Motif, 39064) An h− reporter strain with “green” and “orange” at the ura4 locus was added per ChIP sample. Immune complexes were collected (natMX marked) and “red” at the leu1 locus (hygMX marked) was with Protein A Dynabeads (Thermo Fisher) for all ChIP samples crossed to a 408 strain subset of the Bioneer haploid deletion li- except the anti-Flag ChIP samples, which were collected with brary (kanMX marked). Crosses were performed as described (Ver- Protein G Dynabeads (Thermo Fisher). DNA was quantified by rier et al. 2015; Barrales et al. 2016) with limited modifications. RT-qPCR, and percentage IP (ChIP DNA/input DNA) was calcu- Briefly, crosses were arrayed onto SPAS plates using a RoToR lated as described (Greenstein et al. 2018). HDA colony pinning robot (Singer) and mated for 4 d at room temperature. The plates were incubated for 4 d at 42°C following mating to remove haploid and diploid cells, retaining spores. Re- ChIP-seq sample and library preparation sultant spores were germinated on YES medium with added Sample preparation and ChIP prior to sequencing was performed hygromycin B, G418, and nourseothricin for selection of both re- essentially as described (Greenstein et al. 2018) with the follow- porter loci and the appropriate gene deletion. The resultant colo- ing modifications: 50 mL of cells was grown to OD = 0.6–0.8 over- nies were passaged into liquid YES and grown overnight for flow night from OD = 0.025. Biological duplicate samples were cytometry as described above. In the morning, cells were diluted generated for WT, biological triplicate samples were generated again into YES medium and grown 4–6 h at 32°C prior to analysis for Δepe1, and four biological samples were generated for Δepe1Δ- via flow cytometry. set1 genotypes. Based on OD measurements, 300 × 106 cells per sample were fixed and processed for ChIP. Shearing was per- formed with 20 cycles of 30 sec on/30 sec rest. Samples were RNA extraction and quantification not precleared. Sonication efficiency was determined for each Cells from log phase cultures or FACS-sorted cells were pelleted, sample and only samples where DNAs averaged 200–300 bp and supernatant was decanted and flash-frozen in liquid nitrogen. were used. Chromatin was split into two samples after 8% was Pellets were stored at −80°C. RNA extraction was performed as set aside as input. Three microliters of H3K9me2 (Abcam,1220) described (Greenstein et al. 2018). cDNA synthesis was per- or H3K4me3 (Active Motif, 39159) antibodies was added per formed with either SuperScript RTIII or IV (Invitrogen) and an tube and incubated overnight at 4°C with rotation. (Only oligo dT primer (Fig. 2C; Supplemental Figs. S1A, S3B, S4H) or Su- H3K9me2 ChIP was performed for Δset1 strains. The absence of perScript RTIV (Invitrogen) and random hexamers (Supplemental H3K4me3 was validated by ChIP qPCR in Supplemental Fig. Figs. S4A, S7C) via the manufacturer’s protocol. cDNA samples S2A.) Immune complexes were collected with 30 μL of twice- were quantified by RT-qPCR as described (Greenstein et al. washed Protein A Dynabeads (Invitrogen) for 3 h at 4°C. Beads 2018). Values from cDNA targets were normalized to act1 or were washed as above with the exception that the wash buffer pyk1. Samples in Supplemental Figures S1A, S4H, and S7C step was performed twice. Following incubation for 20 min at were normalized to the target/actin value for the Δclr4 strain of 70°C, DNA was eluted in 100 μL of TE + 1%SDS and the beads a matched background. For Supplemental Figure S4A (left), given were washed and eluted a second time with 100 μLofTE+1% that signal from act1p-driven “red” increases by ∼50% in Δset1 SDS + 5 μL of 20 mg/mL Proteinase K (Roche). Following over- backgrounds, the target/actin values in Δset1 samples were mul- night incubation at 65°C, ChIP and input samples were purified tiplied by the mean ratio Δset1/WT of act1p driven “red” signal using Machery Nagel PCR cleanup kit. Library preparation for se- from the four WT and mutant pairs in Supplemental Figure S4A quencing was performed as described (Inada et al. 2016; Parsa (right). This adjusts the normalization for the up-regulation of ac- et al. 2018). Samples were sequenced on a HiSeq 4000 platform tin observed in this background. (Illumina) with a single-end 50 run.

Chromatin immunoprecipitation ChIP-seq data analysis Chromatin Immunoprecipitation (ChIP) followed by qPCR was Sliding window quality filtering and adapter trimming were car- performed essentially as described (Greenstein et al. 2018) with ried out using Trimmomatic 0.38 (Bolger et al. 2014) before the

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Set1 constrains facultative heterochromatin reads were aligned to the S. pombe genome (Wood et al. 2002) ing protein (MBP) tags. Full-length Clr4 was expressed from a with Bowtie2 2.3.4.2 (Langmead and Salzberg 2012) using stan- previously described vector (Al-Sady et al. 2013). Proteins were dard end-to-end sensitive alignment. Indexed bam files were gen- expressed as described (Al-Sady et al. 2013) except that for Clr4- erated using SAMtools 1.9 (Li et al. 2009) “view,”“sort,” and SET and full-length Clr4 LB was substituted for 2XYT medium

“index” functions. Combined input files and WT H3K9me2 supplemented with 10 μM ZnSO4. Lysis and Talon affinity resin ChIP files were generated with SAMtools “merge” function for purification (Takara Bio) and size exclusion chromatography were use in normalization. Input or WT normalized signal tracks essentially as described (Al-Sady et al. 2013). Lysis buffer was 100 were generated using the MACS2 version 2.1.1.20160309 (Zhang mM HEPES (pH 7.5), 300 mM NaCl, 10% glycerol, 7.5 mM imid- et al. 2008b; Feng et al. 2012) callpeak function to generate reads azole, 0.5% Triton-X100, 1 mM β-mercaptoethanol, and protease per million normalized bedGraph files with the following inhibitors. For Clr4-SET and full-length Clr4, Triton was substi- flags: -g 1.26e7 ‐‐nomodel ‐‐extsize 200 ‐‐keep-dup auto -B ‐‐SPMR tuted for 0.01% Igepal NP-40. After final size exclusion chroma- -q 0.01. The resulting pileup was normalized with the bdgcmp tography, Clr4-CD was eluted into FP storage buffer (20 mM function via the fold enrichment method (m –FE). The resulting HEPES at pH 7.5, 100 mM KCl, 10% glycerol, 5 mM β-mercaptoe- normalized signal track files were trimmed back to the length thanol). Clr4-SET and full-length Clr4 were eluted into Clr4 stor- of the genome and converted to bigwig format using UCSCtools age buffer (100 mM Tris at pH 8.5, 100 mM KCl, 10% glycerol, 1 bedClip and bedGraphToBigWig functions. BigWig files were im- mM MgCl2, 20 µM ZnSO4, 10 mM β-mercaptoethanol). All pro- ported into R 3.5.1 with rtracklayer 1.40.6 (Lawrence et al. 2009). teins were flash-frozen and stored at −80°C. Protein concentra- The genome was divided into 25-bp bins and the average enrich- tions were determined by Sypro Ruby (Bio-Rad) gel staining ment value per bin was calculated using the tileGenome and bin- against a BSA standard curve and for Clr4-CD and Clr4-SET nedAverage functions of GenomicRanges 1.32.7 (Lawrence et al. were verified by UV absorption at 280 nm using the theoretical 2013). Gene annotations were imported from PomBase (Lock extinction coefficient (ExPasy ProtParam) 88,810 cm−1M−1 et al. 2019) and converted to genomic coordinates with the make- and 98,210 cm−1M−1 for Clr4-CD and Clr4-SET, respectively. TxDbFromGFF function from GenomicFeatures 1.32.3 (Law- rence et al. 2013). Finally, mean and confidence interval per each genotype were generated during signal track plotting using Fluorescence polarization assay the DataTrack command from Gviz 1.24.0 (Hahne and Ivanek Fluorescence polarization assay for binding of Clr4-CD or 2016). For the P-value track, reads for H3K9me2 ChIP-seq in full-length Clr4 to H3 tail peptides was performed as described each isolate of each strain were extended to 200 bp and counted (Canzio et al. 2013). Ten nanomolar H3 tail peptide with into sliding 150-bp windows beginning every 30 bp using the win- K4me0K9me0 (unmodified), K4me0K9me3, or K4me3K9me3 dowCounts function from R package csaw 1.18.0 (Lun and Smyth modifications (GenScript) was used as probe. Reactions were per- 2016). Global background was determined from 5-kb bins and a formed in FP buffer (20 mM HEPES at pH 7.5, 100 mM KCl, 10% filter of 1.7 times the global average was applied with the filter- glycerol, 0.01%–0.1% NP-40 substitute) and incubated for 20 min Windows function and subsetting. Composition bias was correct- at room temperature prior to measurement. Fluorescence polari- ed using the TMM method via the normFactors and asDGEList zation measurements and data analysis including fitting of curves functions and then dispersion was calculated via estimateDisp were performed as described (Canzio et al. 2013). function before a generalized linear model based on genotype was fit with glmQLfit. P-values result from testing a contrast between Δepe1Δset1 and Δepe1 based on the fitted model and Histone methyltransferase assay summarizing the per window P-values over 300-bp or 1200-bp bins. P-values were interpreted as colors based on the specified Multiple turnover kinetic assays were performed as described (Al- ranges and added to the signal track plots with the Annotation- Sady et al. 2013) with the following modifications. Reactions con- – Track command from Gviz. Peaks were called with epic2 0.0.14 tained 100 µM cold SAM (disulfate tosylate; Abcam) and 10 15 3H – (Stovner and Saetrom 2019) with the following flags: ‐‐effective- µM SAM tracer (55 75 Ci/mmol; PerkinElmer) and were incu- genome-fraction 0.999968 -bin 200 -g 3 -fs 200 -fdr 0.05. Regions bated with 1 µM Suv39/Clr4-SET or full-length and varying – of known heterochromatin formation were imported from a pre- amounts of biotinylated H3(1 20) peptide with K4me0 (unmodi- viously curated list (Parsa et al. 2018). Regions were extended by fied), K4me2, or K4me3 (GenScript). Reactions were performed at – 10 kb on each side to account for differences in coordinates that 30°C in Clr4 reaction buffer (100 120 mM Tris at pH 8.5, 100 mM may exist for different genome assemblies, as well as variable KCl, 10% glycerol, 1 mM MgCl2, 20 µM ZnSO4,10mM β spreading. Peaks and known regions were plotted using Gviz -cmercaptoethanol). (Hahne and Ivanek 2016). For the global analysis comparison be- tween Δepe1Δset1 and Δepe1 genotypes, the average value per Licor Western blot 300-bp window for the input normalized H3K9me2 ChIP-seq was computed using deeptools2 3.1.3 (Ramírez et al. 2016) func- For Western blot lysate method 2, whole-cell total protein ex- tion multiBigWigSummary. The counts per bin output file was tracts were prepared as described (Al-Sady et al. 2016). For anti- read into R 3.6.0 and the mean value for each genotype was com- Flag Western blot lysate method 1, pellets from 1 mL of saturated puted per bin. The log2 ratio of the Δepe1Δset1 genotype average overnight cultures were flash-frozen, then resuspended in 10% over the Δepe1 genotype average was computed for each bin that trichloro-acetic acid, mixed by vortexing, and then incubated had H3K9me2 signal above a threshold of 1.5 times the global av- for 10 min on ice. The precipitate was washed once with cold ac- erage calculated in the same manner as for P-value track. etone and the pellet was air-dried and then resuspended in 40 µL of Tris/HCl (pH 8) with 200 µL of 2× Laemmli sample buffer. Four-hundred microliters of 0.5-mm glass beads was added per tube, and each sample was mixed in a platform vortexer twice Clr4 purification for 60 sec. The bottom of the tube was then pierced with a 26G The chromodomain of Clr4 (residues 6–64, Clr4-CD) and SET needle and the supernatant was recovered into another tube by domain (residues 192–490, Clr4-SET) were each cloned into Mac- centrifugation. Prior to loading the gel all samples were boiled roLab vector 14C containing N-terminal 6xHis and maltose-bind- for 10 min and then centrifuged at >10,000g for 2 min to remove

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Greenstein et al. insoluble material. Western blot was performed as described (Al- heterochromatin. Nat Struct Mol Biol 20: 547–554. doi:10 Sady et al. 2016) and the following primary antibodies were used .1038/nsmb.2565 H3K4me2 (Active Motif, 39141), H3K4me3 (Active Motif, Ayoub N, Goldshmidt I, Lyakhovetsky R, Cohen A. 2000. A 39159), anti-GAPDH (Thermo Scientific, MA5-15738) and anti- fission yeast repression element cooperates with centro- Flag M2 (Sigma). H3K4me2/3 blots were coincubated with anti- mere-like sequences and defines a mat silent domain boun- GAPDH antisera and followed by both secondary antibodies as dary. Genetics 156: 983–994. described (Al-Sady et al. 2016). For the anti-Flag Western blot, giv- Ayoub N, Noma K, Isaac S, Kahan T, Grewal SIS, Cohen A. 2003. en the size difference between GAPDH and Set1, the membrane A novel jmjC domain protein modulates heterochromatiza- was cut between the 50- and 75-kDa bands on the ladder. The tion in fission yeast. Mol Cell Biol 23: 4356–4370. doi:10 larger half was incubated with anti-Flag 1° and then antimouse .1128/MCB.23.12.4356-4370.2003 2° while the smaller half was incubated separately with anti- Barrales RR, Forn M, Georgescu PR, Sarkadi Z, Braun S. 2016. GAPDH 1° and then antimouse 2°. Control of heterochromatin localization and silencing by the nuclear membrane protein Lem2. Genes Dev 30: 133–148. Bell AC, Felsenfeld G. 1999. Stopped at the border: boundaries Acknowledgments and insulators. Curr Opin Genet Dev 9: 191–198. doi:10 .1016/S0959-437X(99)80029-X We thank Hiten D Madhani for the generous gifts of strains and Binda O, LeRoy G, Bua DJ, Garcia BA, Gozani O, Richard S. 2010. reagents and use of laboratory equipment. Additionally, we thank Trimethylation of histone H3 lysine 4 impairs methylation of Sandra Catania, Michael McManus, Sy Redding, and Kieran Mace histone H3 lysine 9: regulation of lysine methyltransferases by for helpful discussions on data acquisition, analysis, and interpre- physical interaction with their substrates. Epigenetics 5: 767– tation. This work was supported by grants from the National In- 775. doi:10.4161/epi.5.8.13278 stitutes of Health (NIH; DP2GM123484) and the University of Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible California at San Francisco Program for Breakthrough Biomedical trimmer for Illumina sequence data. Bioinformatics 30: Research (partially funded by the Sandler Foundation) to B.A.-S., 2114–2120. doi:10.1093/bioinformatics/btu170 and the ARCS Foundation Scholarship and Hooper Graduate Fel- Braun S, Garcia JF, Rowley M, Rougemaille M, Shankar S, Mad- lowship to R.A.G. R.A.G. was additionally supported by an NIH hani HD. 2011. The Cul4-Ddb1Cdt2 ubiquitin ligase inhibits grant to attend the Cold Spring Harbor Laboratory course Statis- invasion of a boundary-associated antisilencing factor into tical Methods for Functional Genomics. We acknowledge the heterochromatin. Cell 144: 41–54. doi:10.1016/j.cell.2010.11 course instructors for their helpful comments on this work, in .051 particular Sean Davis for insightful suggestions on statistical Brower-Toland B, Wacker DA, Fulbright RM, Lis JT, Kraus WL, analysis of the screen hits. This work was supported by grants Wang MD. 2005. Specific contributions of histone tails and awarded to S.B. from the German Research Foundation (BR their acetylation to the mechanical stability of nucleosomes. 3511/2-1) and the European Union Network of Excellence J Mol Biol 346: 135–146. doi:10.1016/j.jmb.2004.11.056 EpiGeneSys (HEALTH-2010-257082). S.B. is Member of the Bühler M, Verdel A, Moazed D. 2006. Tethering RITS to a nascent Collaborative Research Center 1064 funded by the German Re- transcript initiates RNAi- and heterochromatin-dependent search Foundation and acknowledges infrastructure support. gene silencing. Cell 125: 873–886. doi:10.1016/j.cell.2006.04 J.E.B. is a Natural Sciences and Engineering Research Council .025 of Canada postdoctoral fellow. Flow cytometry and FACS data were generated in the University of California at San Francisco Bühler M, Spies N, Bartel DP, Moazed D. 2008. TRAMP-mediat- Parnassus Flow Cytometry Core, which is supported by Diabetes ed RNA surveillance prevents spurious entry of RNAs into Research Center (DRC) grants NIH P30 DK063720 and NIHS10 the Schizosaccharomyces pombe siRNA pathway. Nat Struct – 1S10OD021822-01. Mol Biol 15: 1015 1023. doi:10.1038/nsmb.1481 Author contributions: R.A.G., B.A.-S., R.R.B., and S.B. designed Buratowski S, Kim T. 2010. The role of cotranscriptional histone – experiments. 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GENES & DEVELOPMENT 19 ERRATUM

Genes & Development 34: 99–117 (2020)

Erratum: Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

R.A. Greenstein, Ramon R. Barrales, Nicholas A. Sanchez, Jordan E. Bisanz, Sigurd Braun, and Bassem Al-Sady

In the above-mentioned article, a statement regarding the availability of the raw sequencing data sets (GEO accession no. GSE140067) was inadvertently omitted. This information has now been added to the article online under the heading Data Sets. doi: 10.1101/gad.342006.120

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Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

R.A. Greenstein, Ramon R. Barrales, Nicholas A. Sanchez, et al.

Genes Dev. published online December 5, 2019 Access the most recent version at doi:10.1101/gad.328468.119

Supplemental http://genesdev.cshlp.org/content/suppl/2019/12/03/gad.328468.119.DC1 Material

Related Content Erratum: Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability R.A. Greenstein, Ramon R. Barrales, Nicholas A. Sanchez, et al. Genes Dev. August , 2020 34: 1106

Published online December 5, 2019 in advance of the full issue.

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